The ventral tegmental area (VTA) controls the motivation to pursue rewards and avoid harmful stimuli. Within the last decade it has been shown that the VTA is populated by neurons that release the neuromodulator dopamine, neurons that release the inhibitory neurotransmitter GABA, and neurons that release the excitatory neurotransmitter glutamate. Based on the use of optogenetic techniques to manipulate dopamine, GABA, or glutamate neurons, current models of VTA function postulate that reward and aversion-based motivation is mediated by heterogeneous types of VTA neurons and their discrete neuronal networks. However, we have recently discovered an unanticipated type of neuron in the VTA, those that release both glutamate and GABA. The discovery of the VTA glutamate-GABA neurons redefines the GABA-releasing and glutamate-releasing VTA cell-types by multiple genetic features: release of glutamate without GABA (glutamate-only neurons), release of GABA without glutamate (GABA-only neurons), or release of both (glutamate-GABA neurons). While optogenetic studies have shown roles of glutamate-releasing or GABA-releasing neurons in motivated behavior, the redefinition of VTA glutamate and GABA-releasing neurons into three subtypes indicates that our understanding of the VTA cell-type specific roles in motivated behavior is incomplete. We will use novel intersectional and subtractive genetic and viral tools to identify the neuronal networks and roles in reward- or aversion-mediated behavior of VTA glutamate-GABA, glutamate-only, and GABA-only neurons and circuits. In Aim 1, we will comprehensively map the brain-wide inputs and outputs of VTA glutamate-GABA, glutamate- only, and GABA-only neurons using cell-type specific anterograde tracing and monosynaptic retrograde tracing with a modified rabies virus. In Aim 2, using the genetically encoded calcium indicator GCaMP6 we will identify the neuronal activity patterns of VTA glutamate-GABA, glutamate-only, and GABA-only neurons, as well as the neuronal activity patterns of major afferents that target each genetically separate VTA cell-type, in response to rewarding and aversive stimuli and their predictors. Finally, we will optogenetically stimulate or inhibit VTA glutamate-GABA, glutamate-only, and GABA-only neurons and circuits to define their causal contributions towards motivated behavior. Preliminary data support the hypothesis that VTA glutamate-GABA, glutamate- only, and GABA-only neurons have distinct neuronal networks and motivational functions. Together, these studies will comprehensively redefine the motivational functions of VTA neurons and their individual circuits. Results will provide new directions to alleviate reward and aversion based disorders such as addiction.